An intense interest in the relationship between oxidative stress and diseases has characterized the last two decades since the observation that oxidative modification of low-density lipoprotein (LDL) leads to foam cell formation and atherosclerosis. Convincing evidence exists that, by opposing the reactive oxygen species (ROS) mediated processes (i.e., lipid peroxidation and other direct hazardous effects) in the body, anti-reactive oxygen-species (Anti-ROS)-antioxidant compounds exert beneficial effects in atherosclerosis and other pathologies involving oxidative stress and free radical injury.
The term “oxidative stress” is commonly used in reference to biological systems as a means to characterize the total burden of potentially harmful reactive oxygen species that are present in tissues as a consequence of routine cellular oxidative metabolism of both endogenous and exogenous compounds. The term itself is a misnomer because many of the chemical reactions that contribute to oxidative-stress are not oxidative in nature. For example, although it usually fictions as a reducing agents the production of superoxide anion in biological systems is often described as a type of oxidative stress. To initiate lipid peroxidation, superoxide must first be converted to another radical species such as the hydroxyl radical via dismutation to hydrogen peroxide, followed by reduction of hydrogen peroxide to hydroxyl radical via the Fenton reaction. [1]. Others may define oxidative stress as the production of free radical species in vivo, though, a number of species associated with oxidative stress either are not radicals or radical species that are not inherently deleterious.
Endothelial cell smooth muscle cells, and monocytes all produce superoxide anion and other ROS under physiological conditions.
The recent discovery of the central role played by the endothelium-derived relaxing factor (EDRF), in control of blood flow and fluidity through the generation of biochemical species that inhibit platelet activation and deposition, prevent thrombus formation, and limit inappropriate vasoconstriction. initiated a series of studies defining the cardinal impact of reactive oxygen species on the activity of EDRF.
EDRF identified as NO or a closely related redox form of NO (i.e., S-nitrosothiol), acts to dilate vascular smooth muscle and inhibit platelet adhesion by activating guanylyl cyclase (GC) and increasing intracellular cyclic-3′,5′-guanosine monophosphate (cGMP) [2,3]. EDRF is constitutively produced and also released from endothelial cells via receptor-mediated mechanisms after exposure to a number of clinically relevant agonists [4]. In addition to EDRF, the vascular endothelium produces a number of vasoactive substrates [5] which act in concert with EDRF to mediate endothelial control of vascular tone and platelet and monocyte activity.
The mechanisms by which superoxide and other ROS may contribute to abnormalities in EDRF action are diverse. Superoxide may react destructively with NO and limit the biological activity of EDRF [6]. Superoxide production may lead to formation of hydroxyl radicals which may be cytotoxic to endothelial cells [7] through direct peroxidation of lipids and proteins. Hydrogen peroxide is formed during the dismutation of superoxide and may, in addition to peroxynitrite, also oxidize available free and proteinous thiol groups that may be important for EDRF action. Hydrogen peroxide is a principal mediator of neutrophile-induced injury to endothelial cells and its effects on other physiological functions are complex, although oxidation of intracellular thiols is a likely mechanism for these effects because pretreatment with dithiothreitol prevents H2O2 mediated cell dysfunction. Hydroxyl radicals have been implicated in tissue damage resulting from repelfusion injury and inflammation, and may be present in athereosclerotic lesions [8]. Organic peroxyl radicals have been implicated in a wide variety of biochemical processes, and these species combine readily with NO leading to the formation of peroxynitrite.
In addition to inactivation of EDRF, oxidative depletion of vital thiol groups may be the mechanism through which ROS exert cell toxicity and dysfunction.
Ample evidence exists that links biological thiols to EDRF action and metabolism. NO is a reactive molecule that readily combines with a number of biochemical species producing a variety of derivative oxides of nitrogen. These derivatives are themselves reactive and can form adducts with readily available sulfhydryl species under physiological conditions producing stable, biologically active S-nitrosothiols that possess biochemical, vasorelaxant and platelet inhibitory properties both in vitro and in vivo [9, 10]. Because S-nitrosothiols possess properties reminiscent of EDRF, many have speculated that EDRF may be a nitrosothiol rather than authentic NO [3].
Thiol availability in mediating the effects of endogenous NO and of exogenous NO-donors (i.e., organic nitrates) is of critical importance [24]. It was demonstrated that the redox-sensitive extra/intracellular thiol content may have critical implications, not only for EDRF (NO) action and metabolism, but also regarding the mechanisms by which anti-ROS thiol-containing compounds may prove useful in preserving normal cellular function as well as preventing/reversing pathological conditions involving NO, thiols and ROS.
The body is endowed against the deleterious effects of ROS, with a number of antioxidant defense mechanisms, which may be divided into three major groups. The first group, enzymatic antioxidants, represents the main form of intracellular antioxidant defenses and mainly includes SOD, catalase, and glutathione peroxidase. The second group, nonenzymatic protein antioxidants, is primarily found in plasma and is mainly represented by GSH, and some proteins such as transferrin albumin, and ceruloplasmin, which also has enzymatic (ferroxidase) activity. Finally, the nonenzymatic low molecular weight antioxidants are found in plasma, extracellular and intracelluar fluids, lipoproteins, and cell membranes. This group of antioxidants may be further subdivided into water-soluble (i.e., GSH, uric and ascorbic acids, and bilirubin) and lipid-soluble antioxidants which are localized to cell membranes and to lipoproteins and include α-tocopherol, β-carotene, and ubiquinol 10. Other endogenous low molecular weight species present in plasma and extracellular fluids also have antioxidant properties including phenolic estrogens, thyroxin and catecholamines.
A possible mechanism of antioxidant-mediated preservation of cellular function is decreased oxidative modification of LDL. However, recent evidence suggests that both water- and lipid-soluble antioxidants may have important physiological effects that are not directly related to the protection of LDL-against oxidation in vivo. Although these alternative effects of antioxidants may not bear directly on EDRF action, they have the potential to influence processes that are known to impair redox-dependent enzymatic and non-enzymatic metabolic processes. One may also speculate that the antioxidant activity of agents like vitamin E and β-carotene, may reflect the free-radical-scavenging characteristics of these agents vis-a-vis superoxide anion or hydroxyl radicals, either directly or via modulation of enzymes action. However, because they lack the possibility to exist in an equilibrium between die two possible forms [disulfide (oxidized) <-> thiols (reduced)] when overdosed, vitamin E, as well as most other currently available antioxidants may adversely affect the course of the disease for which they are indicated [12].
Thiols are more central to cellular antioxidant defense mechanisms than any other existing antioxidant present in the cell (i.e. endothelium, brain, skin and other tissues). However, thiol antioxidants that are effective in vitro, may not be effective in vivo. For example, the thiol antioxidant GSH would seem an ideal candidate for treating endothelial dysfunction and many other diseases involving oxidative stress. Unfortunately, GSH is not absorbed from the diet or through the skin. N-Acetylcysteine (NAC), which provides cysteine for GSH synthesis, and which is readily absorbed and transported is an alternative. However, side effects including nausea vomiting, and diarrhea greatly limit its clinical effectiveness for enteral administration and its extreme instability limits its topical administration [13].
Thus, very few successful pharmacological intervention strategies are currently available for the treatment of endothelial dysfunction and other pathologies involving oxidative stress and free radical injury. Vitamin E, vitamin C, probucol and β-carotene constitute most of antioxidants currently applied. Unfortunately, however, none of these agents by itself (or when combined with others) can adequately address cellular (i.e., skin or endothelial) dysfunction and other oxidative stress-mediated pathologies. Because of their ‘mode’ of action, tissue uptake and other relevant characteristics, all currently available antioxidants can (if at all!) only indirectly affect EDRF metabolism and action act only on certain ROS, and adversely affect the course of the disease if incorrectly dosed.
Lipoic acid (LA), is frequently referred to as “a universal antioxidant” [14]. LA, as lipoamide, has long been known for its role in oxidative metabolism as an essential cofactor in mitochondrial -keto acid dehydrogenase complexes, which was previously thought to be its only role LA is readily taken up by a variety of cells and tissues and is rapidly reduced to their sulfhydryl (dithiol)-form (the dihydroform), as both in vitro and in vivo [15,16]. The reducing power for this comes from both NADH and NADPH [17].
Numerous studies have demonstrated that both LA and DHLA are antioxidants [18,20]. LA scavenges hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also chelates iron, copper, and other transition metals. In addition to those species (including transition metals) acted upon by LA, DHLA scavenges superoxide radicals and peroxyl radicals For example, LA has been shown to modulate cellular reducing equivalent and thus favorably affect complications of diabetes and ischemic injury [17]. LA was also shown to protect against ROS-mediated brain damage following cerebral ischemia in various animal models [21]. It protects against aminoglycoside-induced nephrotoxicity [22]. Both LA and DHLA were shown to protect against peroxynitrite-dependent tyrosine nitration and alpha I-antiproteinase inactivation [23]. In the working heart model of ischemia-reperfusion, LA (especially the R-enantiomer) has been shown to enhance the aortic flow during reoxygenation [24]. In addition, because of its bioconversion to DHLA, administration of LA has been shown to also regenerate other endogamous antioxidants. Current evidence indicates that DHLA can reduce GSSG to GSH, dehydroascorbate and semidehydroascorbyl radical, and ubiquitione, all of which can contribute to vitamin E regeneration from its oxidized form, as well as to reduce thioredoxin [25].
Studies employing diverse types of thiols have been carried out both in vivo and in vitro. None of the studies involving thiols have ever evaluated the role of the dithiol α-lipoic acid (LA, thioctic acid, 1,2-dithiolane-3-valeric acid, 6,8dithiooctanoic acid) or analogs thereof in EDRF or NO-donors action as well as in other pathologies including senescence-mediated wrinkle formation of skin in general, and of facial skin in particular.